Methods for treatment of hepatocellular carcinoma using 3-(4-amino-1-OXO-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione

ABSTRACT

Methods of treating, preventing and/or managing cancer as well as and diseases and disorders associated with, or characterized by, undesired angiogenesis are disclosed. Specific methods encompass the administration of an immunomodulatory compound alone or in combination with a second active ingredient. The invention further relates to methods of reducing or avoiding adverse side effects associated with chemotherapy, radiation therapy, hormonal therapy, biological therapy or immunotherapy which comprise the administration of an immunomodulatory compound. Pharmaceutical compositions, single unit dosage forms, and kits suitable for use in methods of the invention are also disclosed.

This application is a continuation application of U.S. patentapplication No. 10/438,213, filed May 15, 2003 now U.S. Pat. No.7,968,569, presently pending, which claims the benefit of U.S.provisional application nos. 60/380,842, filed May 17, 2002, and60/424,600, filed Nov. 6, 2002, the entireties of which are incorporatedherein by reference.

1. FIELD OF THE INVENTION

This invention relates to methods of treating, preventing and/ormanaging specific cancers, and other diseases including, but not limitedto, those associated with, or characterized by, undesired angiogenesis,by the administration of one or more immunomodulatory compounds alone orin combination with other therapeutics. In particular, the inventionencompasses the use of specific combinations, or “cocktails”, of drugsand other therapy, e.g., radiation to treat these specific cancers,including those refractory to conventional therapy. The invention alsorelates to pharmaceutical compositions and dosing regimens.

2. BACKGROUND OF THE INVENTION 2.1 Pathobiology of Cancer and OtherDiseases

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, or lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia. Theneoplastic lesion may evolve clonally and develop an increasing capacityfor invasion, growth, metastasis, and heterogeneity, especially underconditions in which the neoplastic cells escape the host's immunesurveillance. Roitt, I., Brostoff, J and Kale, D., Immunology,17.1-17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).

There is an enormous variety of cancers which are described in detail inthe medical literature. Examples includes cancer of the lung, colon,rectum, prostate, breast, brain, and intestine. The incidence of cancercontinues to climb as the general population ages, as new cancersdevelop, and as susceptible populations (e.g., people infected with AIDSor excessively exposed to sunlight) grow. A tremendous demand thereforeexists for new methods and compositions that can be used to treatpatients with cancer.

Many types of cancers are associated with new blood vessel formation, aprocess known as angiogenesis. Several of the mechanisms involved intumor-induced angiogenesis have been elucidated. The most direct ofthese mechanisms is the secretion by the tumor cells of cytokines withangiogenic properties. Examples of these cytokines include acidic andbasic fibroblastic growth factor (a,b-FGF), angiogenin, vascularendothelial growth factor (VEGF), and TNF-α. Alternatively, tumor cellscan release angiogenic peptides through the production of proteases andthe subsequent breakdown of the extracellular matrix where somecytokines are stored (e.g., b-FGF). Angiogenesis can also be inducedindirectly through the recruitment of inflammatory cells (particularlymacrophages) and their subsequent release of angiogenic cytokines (e.g.,TNF-α, bFGF).

A variety of other diseases and disorders are also associated with, orcharacterized by, undesired angiogenesis. For example, enhanced orunregulated angiogenesis has been implicated in a number of diseases andmedical conditions including, but not limited to, ocular neovasculardiseases, choroidal neovascular diseases, retina neovascular diseases,rubeosis (neovascularization of the angle), viral diseases, geneticdiseases, inflammatory diseases, allergic diseases, and autoimmunediseases. Examples of such diseases and conditions include, but are notlimited to: diabetic retinopathy; retinopathy of prematurity; cornealgraft rejection; neovascular glaucoma; retrolental fibroplasia; andproliferative vitreoretinopathy.

Accordingly, compounds that can control angiogenesis or inhibit theproduction of certain cytokines, including TNF-α, may be useful in thetreatment and prevention of various diseases and conditions.

2.2 Methods of Treating Cancer

Current cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (see, for example, Stockdale, 1998, Medicine, vol. 3, Rubensteinand Federman, eds., Chapter 12, Section IV). Recently, cancer therapycould also involve biological therapy or immunotherapy. All of theseapproaches pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of a patient or may beunacceptable to the patient. Additionally, surgery may not completelyremove neoplastic tissue. Radiation therapy is only effective when theneoplastic tissue exhibits a higher sensitivity to radiation than normaltissue. Radiation therapy can also often elicit serious side effects.Hormonal therapy is rarely given as a single agent. Although hormonaltherapy can be effective, it is often used to prevent or delayrecurrence of cancer after other treatments have removed the majority ofcancer cells. Biological therapies and immunotherapies are limited innumber and may produce side effects such as rashes or swellings,flu-like symptoms, including fever, chills and fatigue, digestive tractproblems or allergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A majority of cancerchemotherapeutics act by inhibiting DNA synthesis, either directly, orindirectly by inhibiting the biosynthesis of deoxyribonucleotidetriphosphate precursors, to prevent DNA replication and concomitant celldivision. Gilman et al., Goodman and Gilman's: The Pharmacological Basisof Therapeutics, Tenth Ed. (McGraw Hill, New York).

Despite availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks. Stockdale, Medicine, vol. 3, Rubensteinand Federman, eds., ch. 12, sect. 10, 1998. Almost all chemotherapeuticagents are toxic, and chemotherapy causes significant, and oftendangerous side effects including severe nausea, bone marrow depression,and immunosuppression. Additionally, even with administration ofcombinations of chemotherapeutic agents, many tumor cells are resistantor develop resistance to the chemotherapeutic agents. In fact, thosecells resistant to the particular chemotherapeutic agents used in thetreatment protocol often prove to be resistant to other drugs, even ifthose agents act by different mechanism from those of the drugs used inthe specific treatment. This phenomenon is referred to as pleiotropicdrug or multidrug resistance. Because of the drug resistance, manycancers prove refractory to standard chemotherapeutic treatmentprotocols.

Other diseases or conditions associated with, or characterized by,undesired angiogenesis are also difficult to treat. However, somecompounds such as protamine, hepain and steroids have been proposed tobe useful in the treatment of certain specific diseases. Taylor et al.,Nature 297:307 (1982); Folkman et al., Science 221:719 (1983); and U.S.Pat. Nos. 5,001,116 and 4,994,443. Thalidomide and certain derivativesof it have also been proposed for the treatment of such diseases andconditions. U.S. Pat. Nos. 5,593,990, 5,629,327, 5,712,291, 6,071,948and 6,114,355 to D′Amato.

Still, there is a significant need for safe and effective methods oftreating, preventing and managing cancer and other diseases andconditions, particularly for diseases that are refractory to standardtreatments, such as surgery, radiation therapy, chemotherapy andhormonal therapy, while reducing or avoiding the toxicities and/or sideeffects associated with the conventional therapies.

2.3 IMiDs™

A number of studies have been conducted with the aim of providingcompounds that can safely and effectively be used to treat diseasesassociated with abnormal production of TNF-α. See, e.g., Marriott, J.B., et al., Expert Opin. Biol. Ther. 1(4):1-8 (2001); G. W. Muller, etal., Journal of Medicinal Chemistry 39(17): 3238-3240 (1996); and G. W.Muller, et al., Bioorganic & Medicinal Chemistry Letters 8: 2669-2674(1998). Some studies have focused on a group of compounds selected fortheir capacity to potently inhibit TNF-α production by LPS stimulatedPBMC. L. G. Corral, et al., Ann. Rheum. Dis. 58:(Suppl I) 1107-1113(1999). These compounds, which are referred to as IMiDs™ (CelgeneCorporation) or Immunomodulatory Drugs, show not only potent inhibitionof TNF-α but also marked inhibition of LPS induced monocyte IL1β andIL12 production. LPS induced IL6 is also inhibited by immunomodulatorycompounds, albeit partially. These compounds are potent stimulators ofLPS induced IL10. Id. Particular examples of IMiD™s include, but are notlimited to, the substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles described inU.S. Pat. Nos. 6,281,230 and 6,316,471, both to G. W. Muller, et al.

3. SUMMARY OF THE INVENTION

This invention encompasses methods of treating and preventing certaintypes of cancer, including primary and metastatic cancer, as well ascancers that are refractory or resistant to conventional chemotherapy.The methods comprise administering to a patient in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of an immunomodulatory compound, or a pharmaceutically acceptablesalt, solvate, hydrate, stereoisomer, clathrate, or prodrug thereof. Theinvention also encompasses methods of managing certain cancers (e.g.,preventing or prolonging their recurrence, or lengthening the time ofremission) which comprise administering to a patient in need of suchmanagement a prophylactically effective amount of an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof.

In particular methods of the invention, an immunomodulatory compound isadministered in combination with a therapy conventionally used to treat,prevent or manage cancer. Examples of such conventional therapiesinclude, but are not limited to, surgery, chemotherapy, radiationtherapy, hormonal therapy, biological therapy and immunotherapy.

This invention also encompasses methods of treating, managing orpreventing diseases and disorders other than cancer that are associatedwith, or characterized by, undesired angiogenesis, which compriseadministering to a patient in need of such treatment, management orprevention a therapeutically or prophylactically effective amount of animmunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof.

In other methods of the invention, an immunomodulatory compound isadministered in combination with a therapy conventionally used to treat,prevent or manage diseases or disorders associated with, orcharacterized by, undesired angiogenesis. Examples of such conventionaltherapies include, but are not limited to, surgery, chemotherapy,radiation therapy, hormonal therapy, biological therapy andimmunotherapy.

This invention encompasses pharmaceutical compositions, single unitdosage forms, dosing regimens and kits which comprise animmunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and asecond, or additional, active agent. Second active agents includespecific combinations, or “cocktails”, of drugs.

4. BRIEF DESCRIPTION OF FIGURE

FIG. 1 shows a comparison of the effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) and thalidomide in inhibiting the proliferation of multiplemyeloma (MM) cell lines in an in vitro study. The uptake of[³H]-thymidine by different MM cell lines (MM.1S, Hs Sultan, U266 andRPMI-8226) was measured as an indicator of the cell proliferation.

5. DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention encompasses methods of treating,managing, or preventing cancer which comprises administering to apatient in need of such treatment or prevention a therapeutically orprophylactically effective amount of an immunomodulatory compound of theinvention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof.

In particular methods encompassed by this embodiment, theimmunomodulatory compound is administered in combination with anotherdrug (“second active agent”) or method of treating, managing, orpreventing cancer. Second active agents include small molecules andlarge molecules (e.g., proteins and antibodies), examples of which areprovided herein, as well as stem cells. Methods, or therapies, that canbe used in combination with the administration of the immunomodulatorycompound include, but are not limited to, surgery, blood transfusions,immunotherapy, biological therapy, radiation therapy, and other non-drugbased therapies presently used to treat, prevent or manage cancer.

Another embodiment of the invention encompasses methods of treating,managing or preventing diseases and disorders other than cancer that arecharacterized by undesired angiogenesis. These methods comprise theadministration of a therapeutically or prophylactically effective amountof an immunomodulatory compound, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof.

Examples of diseases and disorders associated with, or characterized by,undesired angiogenesis include, but are not limited to, inflammatorydiseases, autoimmune diseases, viral diseases, genetic diseases,allergic diseases, bacterial diseases, ocular neovascular diseases,choroidal neovascular diseases, retina neovascular diseases, andrubeosis (neovascularization of the angle).

In particular methods encompassed by this embodiment, theimmunomodulatory compound is administer in combination with a secondactive agent or method of treating, managing, or preventing the diseaseor condition. Second active agents include small molecules and largemolecules (e.g., proteins and antibodies), examples of which areprovided herein, as well as stem cells. Methods, or therapies, that canbe used in combination with the administration of the immunomodulatorycompound include, but are not limited to, surgery, blood transfusions,immunotherapy, biological therapy, radiation therapy, and other non-drugbased therapies presently used to treat, prevent or manage disease andconditions associated with, or characterized by, undesired angiogenesis.

The invention also encompasses pharmaceutical compositions (e.g., singleunit dosage forms) that can be used in methods disclosed herein.Particular pharmaceutical compositions comprise an immunomodulatorycompound of the invention, or a pharmaceutically acceptable salt,solvate, hydrate, stereoisomer, clathrate, or prodrug thereof, and asecond active agent.

5.1 Immunomodulatory Compounds

Compounds used in the invention include immunomodulatory compounds thatare racemic, stereomerically enriched or stereomerically pure, andpharmaceutically acceptable salts, solvates, hydrates, stereoisomers,clathrates, and prodrugs thereof. Preferred compounds used in theinvention are small organic molecules having a molecular weight lessthan about 1,000 g/mol, and are not proteins, peptides,oligonucleotides, oligosaccharides or other macromolecules.

As used herein and unless otherwise indicated, the terms“immunomodulatory compounds” and “IMiDs™” (Celgene Corporation)encompasses small organic molecules that markedly inhibit TNF-α, LPSinduced monocyte IL1β and IL12, and partially inhibit IL6 production.Specific immunomodulatory compounds are discussed below.

TNF-α is an inflammatory cytokine produced by macrophages and monocytesduring acute inflammation. TNF-α is responsible for a diverse range ofsignaling events within cells. TNF-α may play a pathological role incancer. Without being limited by theory, one of the biological effectsexerted by the immunomodulatory compounds of the invention is thereduction of synthesis of TNF-α. Immunomodulatory compounds of theinvention enhance the degradation of TNF-α mRNA.

Further, without being limited by theory, immunomodulatory compoundsused in the invention may also be potent co-stimulators of T cells andincrease cell proliferation dramatically in a dose dependent manner.Immunomodulatory compounds of the invention may also have a greaterco-stimulatory effect on the CD8+ T cell subset than on the CD4+ T cellsubset. In addition, the compounds preferably have anti-inflammatoryproperties, and efficiently co-stimulate T cells.

Specific examples of immunomodulatory compounds of the invention,include, but are not limited to, cyano and carboxy derivatives ofsubstituted styrenes such as those disclosed in U.S. Pat. No. 5,929,117;1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl) isoindolines and1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such asthose described in U.S. Pat. No. 5,874,448; the tetra substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines(e.g., 4-methyl derivatives of thalidomide and EM-12), including, butnot limited to, those disclosed in U.S. Pat. No. 5,635,517; and a classof non-polypeptide cyclic amides disclosed in U.S. Pat. Nos. 5,698,579and 5,877,200; analogs and derivatives of thalidomide, includinghydrolysis products, metabolites, derivatives and precursors ofthalidomide, such as those described in U.S. Pat. Nos. 5,593,990,5,629,327, and 6,071,948 to D'Amato; aminothalidomide, as well asanalogs, hydrolysis products, metabolites, derivatives and precursors ofaminothalidomide, and substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolessuch as those described in U.S. Pat. Nos. 6,281,230 and 6,316,471;isoindole-imide compounds such as those described in U.S. patentapplication Ser. No. 09/972,487 filed on Oct. 5, 2001, U.S. patentapplication Ser. No. 10/032,286 filed on Dec. 21, 2001, andInternational Application No. PCT/US01/50401 (International PublicationNo. WO 02/059106). The entireties of each of the patents and patentapplications identified herein are incorporated herein by reference.Immunomodulatory compounds of the invention do not include thalidomide.

Other specific immunomodulatory compounds of the invention include, butare not limited to, 1-oxo- and 1,3 dioxo-2-(2,6-dioxopiperidin-3-yl)isoindolines substituted with amino in the benzo ring as described inU.S. Pat. No. 5,635,517 which is incorporated herein by reference. Thesecompounds have the structure I:

in which one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, in particular methyl. Specificimmunomodulatory compounds include, but are not limited to:

1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-6-aminoisoindoline;

1-oxo-2-(2,6-dioxopiperidin-3-yl)-7-aminoisoindoline;

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline; and

1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline.

Other specific immunomodulatory compounds of the invention belong to aclass of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides andsubstituted 2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles, such as thosedescribed in U.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and6,476,052, and International Patent Application No. PCT/US97/13375(International Publication No. WO 98/03502), each of which isincorporated herein by reference. Compounds representative of this classare of the formulas:

wherein R¹ is hydrogen or methyl. In a separate embodiment, theinvention encompasses the use of enantiomerically pure forms (e.g.optically pure (R) or (S) enantiomers) of these compounds.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.Nos. 10/032,286 and 09/972,487, and International Application No.PCT/US01/50401 (International Publication No. WO 02/059106), each ofwhich are incorporated herein by reference. Representative compounds areof formula II:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center.

In specific compounds of formula II, when n is 0 then R¹ is(C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,C(O)R³, C(O)OR⁴, (C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵,(C₁-C₈)alkyl-C(O)OR^(S), C(S)NHR³, or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H or (C₁-C₈)alkyl; and

R³ is (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₅-C₈)alkyl-N(R⁶)₂;(C₀-C₈)alkyl-NH—C(O)O—R⁵; (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵; and the other variables have the samedefinitions.

In other specific compounds of formula II, R² is H or (C₁-C₄)alkyl.

In other specific compounds of formula II, R¹ is (C₁-C₈)alkyl or benzyl.

In other specific compounds of formula II, R¹ is H, (C₁-C₈)alkyl,benzyl, CH₂OCH₃, CH₂CH₂OCH₃, or

In another embodiment of the compounds of formula II, R¹ is

wherein Q is O or S, and each occurrence of R⁷ is independently H,(C₁-C₈)alkyl, benzyl, CH₂OCH₃, or CH₂CH₂OCH₃.

In other specific compounds of formula II, R¹ is C(O)R³.

In other specific compounds of formula II, R³ is(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, (C₁-C₈)alkyl, aryl, or (C₀-C₄)alkyl-OR⁵.

In other specific compounds of formula II, heteroaryl is pyridyl, furyl,or thienyl.

In other specific compounds of formula II, R¹ is C(O)OR⁴.

In other specific compounds of formula II, the H of C(O)NHC(O) can bereplaced with (C₁-C₄)alkyl, aryl, or benzyl.

Still other specific immunomodulatory compounds of the invention belongto a class of isoindole-imides disclosed in U.S. patent application Ser.No. 09/781,179, International Publication No. WO 98/54170, and U.S. Pat.No. 6,395,754, each of which are incorporated herein by reference.Representative compounds are of formula III:

and pharmaceutically acceptable salts, hydrates, solvates, clathrates,enantiomers, diastereomers, racemates, and mixtures of stereoisomersthereof, wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂[X]X₁CH₂CH₂— in which [X]X₁ is—O—, —S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center.

The most preferred immunomodulatory compounds of the invention are4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thecompounds can be obtained via standard, synthetic methods (see e.g.,U.S. Pat. No. 5,635,517, incorporated herein by reference). Thecompounds are available from Celgene Corporation, Warren, N.J.4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (ACTIMID™)has the following chemical structure:

The compound3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(REVIMID™) has the following chemical structure:

Compounds of the invention can either be commercially purchased orprepared according to the methods described in the patents or patentpublications disclosed herein. Further, optically pure compounds can beasymmetrically synthesized or resolved using known resolving agents orchiral columns as well as other standard synthetic organic chemistrytechniques.

As used herein and unless otherwise indicated, the term“pharmaceutically acceptable salt” encompasses non-toxic acid and baseaddition salts of the compound to which the term refers. Acceptablenon-toxic acid addition salts include those derived from organic andinorganic acids or bases know in the art, which include, for example,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embolic acid, enanthic acid, and thelike.

Compounds that are acidic in nature are capable of forming salts withvarious pharmaceutically acceptable bases. The bases that can be used toprepare pharmaceutically acceptable base addition salts of such acidiccompounds are those that form non-toxic base addition salts, i.e., saltscontaining pharmacologically acceptable cations such as, but not limitedto, alkali metal or alkaline earth metal salts and the calcium,magnesium, sodium or potassium salts in particular. Suitable organicbases include, but are not limited to, N,N-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumaine(N-methylglucamine), lysine, and procaine.

As used herein and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,derivatives of immunomodulatory compounds of the invention that comprisebiohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Otherexamples of prodrugs include derivatives of immunomodulatory compoundsof the invention that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties.Prodrugs can typically be prepared using well-known methods, such asthose described in 1 Burger's Medicinal Chemistry and Drug Discovery,172-178, 949-982 (Manfred E. Wolff ed., 5th ed. 1995), and Design ofProdrugs (H. Bundgaard ed., Elselvier, New York 1985).

As used herein and unless otherwise indicated, the terms“biohydrolyzable amide”, “biohydrolyzable ester”, “biohydrolyzablecarbamate”, “biohydrolyzable carbonate”, “biohydrolyzable ureide”,“biohydrolyzable phosphate” mean an amide, ester, carbamate, carbonate,ureide, or phosphate, respectively, of a compound that either: 1) doesnot interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl,acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, andpivaloyloxyethyl esters), lactonyl esters (such as phthalidyl andthiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such asmethoxycarbonyl-oxymethyl, ethoxycarbonyloxyethyl andisopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters,and acylamino alkyl esters (such as acetamidomethyl esters). Examples ofbiohydrolyzable amides include, but are not limited to, lower alkylamides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamatesinclude, but are not limited to, lower alkylamines, substitutedethylenediamines, amino acids, hydroxyalkylamines, heterocyclic andheteroaromatic amines, and polyether amines.

Various immunomodulatory compounds of the invention contain one or morechiral centers, and can exist as racemic mixtures of enantiomers ormixtures of diastereomers. This invention encompasses the use ofstereomerically pure forms of such compounds, as well as the use ofmixtures of those forms. For example, mixtures comprising equal orunequal amounts of the enantiomers of a particular immunomodulatorycompounds of the invention may be used in methods and compositions ofthe invention. These isomers may be asymmetrically synthesized orresolved using standard techniques such as chiral columns or chiralresolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racematesand Resolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., etal., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of CarbonCompounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of ResolvingAgents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind., 1972).

As used herein and unless otherwise indicated, the term “stereomericallypure” means a composition that comprises one stereoisomer of a compoundand is substantially free of other stereoisomers of that compound. Forexample, a stereomerically pure composition of a compound having onechiral center will be substantially free of the opposite enantiomer ofthe compound. A stereomerically pure composition of a compound havingtwo chiral centers will be substantially free of other diastereomers ofthe compound. A typical stereomerically pure compound comprises greaterthan about 80% by weight of one stereoisomer of the compound and lessthan about 20% by weight of other stereoisomers of the compound, morepreferably greater than about 90% by weight of one stereoisomer of thecompound and less than about 10% by weight of the other stereoisomers ofthe compound, even more preferably greater than about 95% by weight ofone stereoisomer of the compound and less than about 5% by weight of theother stereoisomers of the compound, and most preferably greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound. As usedherein and unless otherwise indicated, the term “stereomericallyenriched” means a composition that comprises greater than about 60% byweight of one stereoisomer of a compound, preferably greater than about70% by weight, more preferably greater than about 80% by weight of onestereoisomer of a compound. As used herein and unless otherwiseindicated, the term “enantiomerically pure” means a stereomerically purecomposition of a compound having one chiral center. Similarly, the term“stereomerically enriched” means a stereomerically enriched compositionof a compound having one chiral center.

It should be noted that if there is a discrepancy between a depictedstructure and a name given that structure, the depicted structure is tobe accorded more weight. In addition, if the stereochemistry of astructure or a portion of a structure is not indicated with, forexample, bold or dashed lines, the structure or portion of the structureis to be interpreted as encompassing all stereoisomers of it.

5.2 Second Active Agents

Immunomodulatory compounds can be combined with other pharmacologicallyactive compounds (“second active agents”) in methods and compositions ofthe invention. It is believed that certain combinations worksynergistically in the treatment of particular types of cancer andcertain diseases and conditions associated with, or characterized by,undesired angiogenesis. Immunomodulatory compounds can also work toalleviate adverse effects associated with certain second active agents,and some second active agents can be used to alleviate adverse effectsassociated with immunomodulatory compounds.

One or more second active ingredients or agents can be used in themethods and compositions of the invention together with animmunomodulatory compound. Second active agents can be large molecules(e.g., proteins) or small molecules (e.g., synthetic inorganic,organometallic, or organic molecules).

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. Typical large molecule active agents arebiological molecules, such as naturally occurring or artificially madeproteins. Proteins that are particularly useful in this inventioninclude proteins that stimulate the survival and/or proliferation ofhematopoietic precursor cells and immunologically active poietic cellsin vitro or in vivo. Others stimulate the division and differentiationof committed erythroid progenitors in cells in vitro or in vivo.Particular proteins include, but are not limited to: interleukins, suchas IL-2 (including recombinant IL-II (“rIL2”) and canarypox IL-2),IL-10, IL-12, and IL-18; interferons, such as interferon alfa-2a,interferon alfa-2b, interferon alfa-n1, interferon alfa-n3, interferonbeta-I a, and interferon gamma-I b; GM-CF and GM-CSF; and EPO.

Particular proteins that can be used in the methods and compositions ofthe invention include, but are not limited to: filgrastim, which is soldin the United States under the trade name Neupogen® (Amgen, ThousandOaks, Calif.); sargramostim, which is sold in the United States underthe trade name Leukine® (Immunex, Seattle, Wash.); and recombinant EPO,which is sold in the United States under the trade name Epogen® (Amgen,Thousand Oaks, Calif.).

Recombinant and mutated forms of GM-CSF can be prepared as described inU.S. Pat. Nos. 5,391,485; 5,393,870; and 5,229,496; all of which areincorporated herein by reference. Recombinant and mutated forms of G-CSFcan be prepared as described in U.S. Pat. Nos. 4,810,643; 4,999,291;5,528,823; and 5,580,755; all of which are incorporated herein byreference.

This invention encompasses the use of native, naturally occurring, andrecombinant proteins. The invention further encompasses mutants andderivatives (e.g., modified forms) of naturally occurring proteins thatexhibit, in vivo, at least some of the pharmacological activity of theproteins upon which they are based. Examples of mutants include, but arenot limited to, proteins that have one or more amino acid residues thatdiffer from the corresponding residues in the naturally occurring formsof the proteins. Also encompassed by the term “mutants” are proteinsthat lack carbohydrate moieties normally present in their naturallyoccurring forms (e.g., nonglycosylated forms). Examples of derivativesinclude, but are not limited to, pegylated derivatives and fusionproteins, such as proteins formed by fusing IgG1 or IgG3 to the proteinor active portion of the protein of interest. See, e.g., Penichet, M. L.and Morrison, S. L., J. Immunol. Methods 248:91-101 (2001).

Antibodies that can be used in combination with compounds of theinvention include monoclonal and polyclonal antibodies. Examples ofantibodies include, but are not limited to, trastuzumab (Herceptin®),rituximab (Rituxan®), bevacizumab (Avastin™), pertuzumab (Omnitarg™),tositumomab (Bexxar®), edrecolomab (Panorex®), and G250. Compounds ofthe invention can also be combined with, or used in combination with,anti-TNF-αantibodies.

Large molecule active agents may be administered in the form ofanti-cancer vaccines. For example, vaccines that secrete, or cause thesecretion of, cytokines such as IL-2, G-CSF, and GM-CSF can be used inthe methods, pharmaceutical compositions, and kits of the invention.See, e.g., Emens, L. A., et al., Curr. Opinion Mol. Ther. 3(1):77-84(2001).

In one embodiment of the invention, the large molecule active agentreduces, eliminates, or prevents an adverse effect associated with theadministration of an immunomodulatory compound. Depending on theparticular immunomodulatory compound and the disease or disorder begintreated, adverse effects can include, but are not limited to, drowsinessand somnolence, dizziness and orthostatic hypotension, neutropenia,infections that result from neutropenia, increased HIV-viral load,bradycardia, Stevens-Johnson Syndrome and toxic epidermal necrolysis,and seizures (e.g., grand mal convulsions). A specific adverse effect isneutropenia.

Second active agents that are small molecules can also be used toalleviate adverse effects associated with the administration of animmunomodulatory compound. However, like some large molecules, many arebelieved to be capable of providing a synergistic effect whenadministered with (e.g., before, after or simultaneously) animmunomodulatory compound. Examples of small molecule second activeagents include, but are not limited to, anti-cancer agents, antibiotics,immunosuppressive agents, and steroids.

Examples of anti-cancer agents include, but are not limited to:acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine;simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicinhydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; antiandrogen, prostaticcarcinoma; antiestrogen; antineoplaston; antisense oligonucleotides;aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine;atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid;bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane;buthionine sulfoximine; calcipotriol; calphostin C; camptothecinderivatives; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imatinib (e.g., Gleevec®),imiquimod; immunostimulant peptides; insulin-like growth factor-1receptor inhibitor; interferon agonists; interferons; interleukins;iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemiainhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine;mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide;mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene;molgramostim; Erbitux, human chorionic gonadotrophin; monophosphoryllipid A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; oblimersen (Genasense®);O⁶-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin;SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine;senescence derived inhibitor 1; sense oligonucleotides; signaltransduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate;sodium phenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; translation inhibitors; tretinoin;triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron;turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; vapreotide; variolin B; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Specific second active agents include, but are not limited to,oblimersen (Genasense®), remicade, docetaxel, celecoxib, melphalan,dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum,temozolomide, etoposide, cyclophosphamide, temodar, carboplatin,procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®,taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11,interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A),capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomaldaunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2,GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin,busulphan, prednisone, bisphosphonate, arsenic trioxide, vincristine,doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estramustinesodium phosphate (Emcyt®), sulindac, and etoposide.

5.3 Methods of Treatments and Prevention

Methods of this invention encompass methods of treating, preventingand/or managing various types of cancer and diseases and disordersassociated with, or characterized by, undesired angiogenesis. As usedherein, unless otherwise specified, the term “treating” refers to theadministration of a compound of the invention or other additional activeagent after the onset of symptoms of the particular disease or disorder.As used herein, unless otherwise specified, the term “preventing” refersto the administration prior to the onset of symptoms, particularly topatients at risk of cancer, and other diseases and disorders associatedwith, or characterized by, undesired angiogenesis. The term “prevention”includes the inhibition of a symptom of the particular disease ordisorder. Patients with familial history of cancer and diseases anddisorders associated with, or characterized by, undesired angiogenesisare preferred candidates for preventive regimens. As used herein andunless otherwise indicated, the term “managing” encompasses preventingthe recurrence of the particular disease or disorder in a patient whohad suffered from it, and/or lengthening the time a patient who hadsuffered from the disease or disorder remains in remission.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood born tumors. The term “cancer” refers to disease ofskin tissues, organs, blood, and vessels, including, but not limited to,cancers of the bladder, bone or blood, brain, breast, cervix, chest,colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes,lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis,throat, and uterus. Specific cancers include, but are not limited to,advanced malignancy, amyloidosis, neuroblastoma, meningioma,hemangiopericytoma, multiple brain metastase, glioblastoma multiforms,glioblastoma, brain stem glioma, poor prognosis malignant brain tumor,malignant glioma, recurrent malignant giolma, anaplastic astrocytoma,anaplastic oligodendroglioma, neuroendocrine tumor, rectaladenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectalcarcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma,karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuselarge B-Cell lymphoma, low grade follicular lymphoma, malignantmelanoma, malignant mesothelioma, malignant pleural effusionmesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma,gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneousvasculitis, Langerhans cell histiocytosis, leiomyosarcoma,fibrodysplasia ossificans progressive, hormone refractory prostatecancer, resected high-risk soft tissue sarcoma, unrescectablehepatocellular carcinoma, Waldenstrom's macroglobulinemia, smolderingmyeloma, indolent myeloma, fallopian tube cancer, androgen independentprostate cancer, androgen dependent stage IV non-metastatic prostatecancer, hormone-insensitive prostate cancer, chemotherapy-insensitiveprostate cancer, papillary thyroid carcinoma, follicular thyroidcarcinoma, medullary thyroid carcinoma, and leiomyoma. In a specificembodiment, the cancer is metastatic. In another embodiment, the canceris refractory or resistance to chemotherapy or radiation; in particular,refractory to thalidomide.

As used herein to refer to diseases and conditions other than cancer,the terms “diseases or disorders associated with, or characterized by,undesired angiogenesis”, “diseases or disorders associated withundesired angiogenesis”, and “diseases or disorders characterized byundesired angiogenesis” refer to diseases, disorders and conditions thatare caused, mediated or attended by undesired, unwanted or uncontrolledangiogenesis, including, but not limited to, inflammatory diseases,autoimmune diseases, genetic diseases, allergic diseases, bacterialdiseases, ocular neovascular diseases, choroidal neovascular diseases,and retina neovascular diseases.

Examples of such diseases or disorders associated with undesiredangiogenesis include, but are not limited to, diabetic retinopathy,retinopathy of prematurity, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, proliferative vitreoretinopathy,trachoma, myopia, optic pits, epidemnic keratoconjunctivitis, atopickeratitis, superior limbic keratitis, pterygium keratitis sicca,sjogrens, acne rosacea, phylectenulosis, syphilis, lipid degeneration,bacterial ulcer, fungal ulcer, Herpes simplex infection, Herpes zosterinfection, protozoan infection, Kaposi sarcoma, Mooren ulcer, Terrien'smarginal degeneration, mariginal keratolysis, rheumatoid arthritis,systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,Steven's Johnson disease, periphigoid radial keratotomy, sickle cellanemia, sarcoid, pseudoxanthoma elasticum, Pagets disease, veinocclusion, artery occlusion, carotid obstructive disease, chronicuveitis, chronic vitritis, Lyme's disease, Eales disease, Behcet'sdisease, retinitis, choroiditis, presumed ocular histoplasmosis, Bestsdisease, Stargarts disease, pars planitis, chronic retinal detachment,hyperviscosity syndromes, toxoplasmosis, rubeosis, sarcodisis,sclerosis, soriatis, psoriasis, primary sclerosing cholangitis,proctitis, primary biliary srosis, idiopathic pulmonary fibrosis, andalcoholic hepatitis.

In specific embodiments of the invention, diseases or disordersassociated with undesired angiogenesis do not include congestive heartfailure, cardiomyopathy, pulmonary edema, endotoxin-mediated septicshock, acute viral myocarditis, cardiac allograft rejection, myocardialinfarction, HIV, hepatitis, adult respiratory distress syndrome,bone-resorption disease, chronic obstructive pulmonary diseases, chronicpulmonary inflammatory disease, dermatitis, cystic fibrosis, septicshock, sepsis, endotoxic shock, hemodynamic shock, sepsis syndrome, postischemic reperfusion injury, meningitis, psoriasis, fibrotic disease,cachexia, graft rejection, rheumatoid spondylitis, osteoporosis, Crohn'sdisease, ulcerative colitis, inflammatory-bowel disease, multiplesclerosis, systemic lupus erythrematosus, erythema nodosum leprosum inleprosy, radiation damage, asthma, hyperoxic alveolar injury, malaria,mycobacterial infection, and opportunistic infections resulting fromHIV.

This invention encompasses methods of treating patients who have beenpreviously treated for cancer or diseases or disorders associated with,or characterized by, undesired angiogenesis, but are non-responsive tostandard therapies, as well as those who have not previously beentreated. The invention also encompasses methods of treating patientsregardless of patient's age, although some diseases or disorders aremore common in certain age groups. The invention further encompassesmethods of treating patients who have undergone surgery in an attempt totreat the disease or condition at issue, as well as those who have not.Because patients with cancer and diseases and disorders characterized byundesired angiogenesis have heterogenous clinical manifestations andvarying clinical outcomes, the treatment given to a patient may vary,depending on his/her prognosis. The skilled clinician will be able toreadily determine without undue experimentation specific secondaryagents, types of surgery, and types of non-drug based standard therapythat can be effectively used to treat an individual patient with cancerand other diseases or disorders.

Methods encompassed by this invention comprise administering one or moreimmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, to a patient (e.g., a human) suffering, or likely to suffer,from cancer or a disease or disorder mediated by undesired angiogenesis.

In one embodiment of the invention, an immunomodulatory compound of theinvention can be administered orally and in single or divided dailydoses in an amount of from about 0.10 to about 150 mg/day. In aparticular embodiment,4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™)may be administered in an amount of from about 0.1 to about 1 mg perday, or alternatively from about 0.1 to about 5 mg every other day. In apreferred embodiment,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl-piperidine-2,6-dione(Revimid™) may be administered in an amount of from about 5 to 25 mg perday, or alternatively from about 10 to about 50 mg every other day.

In a specific embodiment,4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™)may be administered in an amount of about 1, 2, or 5 mg per day topatients with relapsed multiple myeloma. In a particular embodiment,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) may be administered initially in an amount of 5 mg/day andthe dose can be escalated every week to 10, 20, 25, 30 and 50 mg/day. Ina specific embodiment, Revimid™ can be administered in an amount of upto about 30 mg/day to patients with solid tumor. In a particularembodiment, Revimid™ can be administered in an amount of up to about 40mg/day to patients with glioma.

5.3.1 Combination Therapy with a Second Active Agent

Specific methods of the invention comprise administering animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, in combination with one or more second active agents, and/or incombination with radiation therapy, blood transfusions, or surgery.Examples of immunomodulatory compounds of the invention are disclosedherein (see, e.g., section 5.1). Examples of second active agents arealso disclosed herein (see, e.g., section 5.2).

Administration of the immunomodulatory compounds and the second activeagents to a patient can occur simultaneously or sequentially by the sameor different routes of administration. The suitability of a particularroute of administration employed for a particular active agent willdepend on the active agent itself (e.g., whether it can be administeredorally without decomposing prior to entering the blood stream) and thedisease being treated. A preferred route of administration for animmunomodulatory compound of the invention is orally. Preferred routesof administration for the second active agents or ingredients of theinvention are known to those of ordinary skill in the art. See, e.g.,Physicians' Desk Reference, 1755-1760 (56^(th) ed., 2002).

In one embodiment of the invention, the second active agent isadministered intravenously or subcutaneously and once or twice daily inan amount of from about 1 to about 1000 mg, from about 5 to about 500mg, from about 10 to about 350 mg, or from about 50 to about 200 mg. Thespecific amount of the second active agent will depend on the specificagent used, the type of disease being treated or managed, the severityand stage of disease, and the amount(s) of immunomodulatory compounds ofthe invention and any optional additional active agents concurrentlyadministered to the patient. In a particular embodiment, the secondactive agent is oblimersen (Genasense®), GM-CSF, G-CSF, EPO, taxotere,irinotecan, dacarbazine, transretinoic acid, topotecan, pentoxifylline,ciprofloxacin, dexamethasone, vincristine, doxorubicin, COX-2 inhibitor,IL2, IL8, IL18, IFN, Ara-C, vinorelbine, or a combination thereof.

In a particular embodiment, GM-CSF, G-CSF or EPO is administeredsubcutaneously during about five days in a four or six week cycle in anamount of from about 1 to about 750 mg/m²/day, preferably in an amountof from about 25 to about 500 mg/m²/day, more preferably in an amount offrom about 50 to about 250 mg/m²/day, and most preferably in an amountof from about 50 to about 200 mg/m²/day. In a certain embodiment, GM-CSF may be administered in an amount of from about 60 to about 500 mcg/m²intravenously over 2 hours, or from about 5 to about 12 mcg/m²/daysubcutaneously. In a specific embodiment, G-CSF may be administeredsubcutaneously in an amount of about 1 mcg/kg/day initially and can beadjusted depending on rise of total granulocyte counts. The maintenancedose of G-CSF may be administered in an amount of about 300 (in smallerpatients) or 480 mcg subcutaneously. In a certain embodiment, EPO may beadministered subcutaneously in an amount of 10,000 Unit 3 times perweek.

In another embodiment, Revimid™ in an amount of about 25 mg/d anddacarbazine in an amount of about from 200 to 1,000 mg/m²/d areadministered to patients with metastatic malignant melanoma. In aspecific embodiment, Revimid™ is administered in an amount of from about5 to about 25 mg/d to patients with metastatic malignant melanoma whosedisease has progressed on treatment with dacarbazine, IL-2 or IFN. In aspecific embodiment, Revimid™ is administered to patients with relapsedor refractory multiple myeloma in an amount of about 15 mg/d twice a dayor about 30 mg/d four times a day in a combination with dexamethasone.

In another embodiment, an immunomodulatory compound is administered withmelphalan and dexamethasone to patients with amyloidosis. In a specificembodiment, an immunomodulatory compound of the invention and steroidscan be administered to patients with amyloidosis.

In another embodiment, an immunomodulatory compound is administered withgemcitabine and cisplatinum to patients with locally advanced ormetastatic transitional cell bladder cancer.

In another embodiment, an immunomodulatory compound is administered incombination with a second active ingredient as follows: temozolomide topediatric patients with relapsed or progressive brain tumors orrecurrent neuroblastoma; celecoxib, etoposide and cyclophosphamide forrelapsed or progressive CNS cancer; temodar to patients with recurrentor progressive meningioma, malignant meningioma, hemangiopericytoma,multiple brain metastases, relapased brain tumors, or newly diagnosedglioblastoma multiforms; irinotecan to patients with recurrentglioblastoma; carboplatin to pediatric patients with brain stem glioma;procarbazine to pediatric patients with progressive malignant gliomas;cyclophosphamide to patients with poor prognosis malignant brain tumors,newly diagnosed or recurrent glioblastoma multiforms; Gliadel® for highgrade recurrent malignant gliomas; temozolomide and tamoxifen foranaplastic astrocytoma; or topotecan for gliomas, glioblastoma,anaplastic astrocytoma or anaplastic oligodendroglioma.

In another embodiment, an immunomodulatory compound is administered withmethotrexate and cyclophosphamide to patients with metastatic breastcancer.

In another embodiment, an immunomodulatory compound is administered withtemozolomide to patients with neuroendocrine tumors.

In another embodiment, an immunomodulatory compound is administered withgemcitabine to patients with recurrent or metastatic head or neckcancer. In another embodiment, an immunomodulatory compound isadministered with gemcitabine to patients with pancreatic cancer.

In another embodiment, an immunomodulatory compound is administered topatients with colon cancer in combination with Arisa®, taxol and/ortaxotere.

In another embodiment, an immunomodulatory compound is administered withcapecitabine to patients with refractory colorectal cancer or patientswho fail first line therapy or have poor performance in colon or rectaladenocarcinoma.

In another embodiment, an immunomodulatory compound is administered incombination with fluorouracil, leucovorin, and irinotecan to patientswith Dukes C & D colorectal cancer or to patients who have beenpreviously treated for metastatic colorectal cancer.

In another embodiment, an immunomodulatory compound is administered topatients with refractory colorectal cancer in combination withcapecitabine, xeloda, and/or CPT-11.

In another embodiment, an immunomodulatory compound of the invention isadministered with capecitabine and irinotecan to patients withrefractory colorectal cancer or to patients with unresectable ormetastatic colorectal carcinoma.

In another embodiment, an immunomodulatory compound is administeredalone or in combination with interferon alpha or capecitabine topatients with unresectable or metastatic hepatocellular carcinoma; orwith cisplatin and thiotepa to patients with primary or metastatic livercancer.

In another embodiment, an immunomodulatory compound is administered incombination with pegylated interferon alpha to patients with Kaposi'ssarcoma.

In another embodiment, an immunomodulatory compound is administered incombination with fludarabine, carboplatin, and/or topotecan to patientswith refractory or relapsed or high-risk acuted myelogenous leukemia.

In another embodiment, an immunomodulatory compound is administered incombination with liposomal daunorubicin, topotecan and/or cytarabine topatients with unfavorable karotype acute myeloblastic leukemia.

In another embodiment, an immunomodulatory compound is administered incombination with gemcitabine and irinotecan to patients with non-smallcell lung cancer. In one embodiment, an immunomodulatory compound isadministered in combination with carboplatin and irinotecan to patientswith non-small cell lung cancer. In one embodiment, an immunomodulatorycompound is administered with doxetaxol to patients with non-small celllung cancer who have been previously treated with carbo/VP 16 andradiotherapy.

In another embodiment, an immunomodulatory compound is administered incombination with carboplatin and/or taxotere, or in combination withcarboplatin, pacilitaxel and/or thoracic radiotherapy to patients withnon-small cell lung cancer. In a specific embodiment, animmunomodulatory compound is administered in combination with taxotereto patients with stage IIIB or IV non-small cell lung cancer.

In another embodiment, an immunomodulatory compound of the invention isadministered in combination with oblimersen (Genasense®) to patientswith small cell lung cancer.

In another embodiment, an immunomodulatory compound is administeredalone or in combination with a second active ingredient such asvinblastine or fludarabine to patients with various types of lymphoma,including, but not limited to, Hodgkin's lymphoma, non-Hodgkin'slymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuselarge B-Cell lymphoma or relapsed or refractory low grade follicularlymphoma.

In another embodiment, an immunomodulatory compound is administered incombination with taxotere, IL-2, IFN, GM-CSF, and/or dacarbazine topatients with various types or stages of melanoma.

In another embodiment, an immunomodulatory compound is administeredalone or in combination with vinorelbine to patients with malignantmesothelioma, or stage IIIB non-small cell lung cancer with pleuralimplants or malignant pleural effusion mesothelioma syndrome.

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of multiple myeloma in combinationwith dexamethasone, zoledronic acid, palmitronate, GM-CSF, biaxin,vinblastine, melphalan, busulphan, cyclophosphamide, IFN, palmidronate,prednisone, bisphosphonate, celecoxib, arsenic trioxide, PEG INTRON-A,vincristine, or a combination thereof.

In another embodiment, an immunomodulatory compound is administered topatients with relapsed or refractory multiple myeloma in combinationwith doxorubicin (Doxil®), vincristine and/or dexamethasone (Decadron®).

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of ovarian cancer such asperitoneal carcinoma, papillary serous carcinoma, refractory ovariancancer or recurrent ovarian cancer, in combination with taxol,carboplatin, doxorubicin, gemcitabine, cisplatin, xeloda, paclitaxel,dexamethasone, or a combination thereof.

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of prostate cancer, in combinationwith xeloda, 5 FU/LV, gemcitabine, irinotecan plus gemcitabine,cyclophosphamide, vincristine, dexamethasone, GM-CSF, celecoxib,taxotere, ganciclovir, paclitaxel, adriamycin, docetaxel, estramustine,Emcyt, or a combination thereof.

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of renal cell cancer, incombination with capecitabine, IFN, tamoxifen, IL-2, GM-CSF, Celebrex®,or a combination thereof.

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of gynecologic, uterus or softtissue sarcoma cancer in combination with IFN, a COX-2 inhibitor such asCelebrex®, and/or sulindac.

In another embodiment, an immunomodulatory compound is administered topatients with various types or stages of solid tumors in combinationwith celebrex, etoposide, cyclophosphamide, docetaxel, apecitabine, IFN,tamoxifen, IL-2, GM-CSF, or a combination thereof.

In another embodiment, an immunomodulatory compound is administered topatients with scleroderma or cutaneous vasculitis in combination withcelebrex, etoposide, cyclophosphamide, docetaxel, apecitabine, IFN,tamoxifen, IL-2, GM-CSF, or a combination thereof.

This invention also encompasses a method of increasing the dosage of ananti-cancer drug or agent that can be safely and effectivelyadministered to a patient, which comprises administering to a patient(e.g., a human) an immunomodulatory compound of the invention, or apharmaceutically acceptable derivative, salt, solvate, clathrate,hydrate, or prodrug thereof. Patients that can benefit by this methodare those likely to suffer from an adverse effect associated withanti-cancer drugs for treating a specific cancer of the skin,subcutaneous tissue, lymph nodes, brain, lung, liver, bone, intestine,colon, heart, pancreas, adrenal, kidney, prostate, breast, colorectal,or combinations thereof. The administration of an immunomodulatorycompound of the invention alleviates or reduces adverse effects whichare of such severity that it would otherwise limit the amount ofanti-cancer drug.

In one embodiment, an immunomodulatory compound of the invention can beadministered orally and daily in an amount of from about 0.1 to about150 mg, and preferably from about 1 to about 50 mg, more preferably fromabout 2 to about 25 mg prior to, during, or after the occurrence of theadverse effect associated with the administration of an anti-cancer drugto a patient. In a particular embodiment, an immunomodulatory compoundof the invention is administered in combination with specific agentssuch as heparin, aspirin, coumadin, or G-CSF to avoid adverse effectsthat are associated with anti-cancer drugs such as but not limited toneutropenia or thrombocytopenia.

In one embodiment, an immunomodulatory compound of the invention can beadministered to patients with diseases and disorders associated with, orcharacterized by, undesired angiogenesis in combination with additionalactive ingredients including but not limited to anti-cancer drugs,anti-inflammatories, antihistamines, antibiotics, and steroids.

In another embodiment, this invention encompasses a method of treating,preventing and/or managing cancer, which comprises administering animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, in conjunction with (e.g. before, during, or after)conventional therapy including, but not limited to, surgery,immunotherapy, biological therapy, radiation therapy, or other non-drugbased therapy presently used to treat, prevent or manage cancer. Thecombined use of the immunomodulatory compounds of the invention andconventional therapy may provide a unique treatment regimen that isunexpectedly effective in certain patients. Without being limited bytheory, it is believed that immunomodulatory compounds of the inventionmay provide additive or synergistic effects when given concurrently withconventional therapy.

As discussed elsewhere herein, the invention encompasses a method ofreducing, treating and/or preventing adverse or undesired effectsassociated with conventional therapy including, but not limited to,surgery, chemotherapy, radiation therapy, hormonal therapy, biologicaltherapy and immunotherapy. One or more immunomodulatory compounds of theinvention and other active ingredient can be administered to a patientprior to, during, or after the occurrence of the adverse effectassociated with conventional therapy.

In one embodiment, an immunomodulatory compound of the invention can beadministered in an amount of from about 0.1 to about 150 mg, andpreferably from about 1 to about 25 mg, more preferably from about 2 toabout 10 mg orally and daily alone, or in combination with a secondactive agent disclosed herein (see, e.g., section 5.2), prior to,during, or after the use of conventional therapy.

In a specific embodiment of this method, an immunomodulatory compound ofthe invention and doxetaxol are administered to patients with non-smallcell lung cancer who were previously treated with carbo/VP 16 andradiotherapy.

5.3.2 Use with Transplantation Therapy

Compounds of the invention can be used to reduce the risk of GraftVersus Host Disease (GVHD). Therefore, the invention encompasses amethod of treating, preventing and/or managing cancer, which comprisesadministering the immunomodulatory compound of the invention, or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof, in conjunction with transplantationtherapy.

As those of ordinary skill in the art are aware, the treatment of canceris often based on the stages and mechanism of the disease. For example,as inevitable leukemic transformation develops in certain stages ofcancer, transplantation of peripheral blood stem cells, hematopoieticstem cell preparation or bone marrow may be necessary. The combined useof the immunomodulatory compound of the invention and transplantationtherapy provides a unique and unexpected synergism. In particular, animmunomodulatory compound of the invention exhibits immunomodulatoryactivity that may provide additive or synergistic effects when givenconcurrently with transplantation therapy in patients with cancer.

An immunomodulatory compound of the invention can work in combinationwith transplantation therapy reducing complications associated with theinvasive procedure of transplantation and risk of GVHD. This inventionencompasses a method of treating, preventing and/or managing cancerwhich comprises administering to a patient (e.g., a human) animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof, before, during, or after the transplantation of umbilical cordblood, placental blood, peripheral blood stem cell, hematopoietic stemcell preparation or bone marrow. Examples of stem cells suitable for usein the methods of the invention are disclosed in U.S. provisional patentapplication No. 60/372,348, filed Apr. 12, 2002 by R. Hariri et al., theentirety of which is incorporated herein by reference.

In one embodiment of this method, an immunomodulatory compound of theinvention is administered to patients with multiple myeloma before,during, or after the transplantation of autologous peripheral bloodprogenitor cell.

In another embodiment, an immunomodulatory compound is administered topatients with relapsing multiple myeloma after the stem celltransplantation.

In another embodiment, an immunomodulatory compound and prednisone areadministered as maintenance therapy to patients with multiple myelomafollowing the transplantation of autologous stem cell.

In another embodiment, an immunomodulatory compound and dexamethasoneare administered as salvage therapy for low risk post transplantation topatients with multiple myeloma.

In another embodiment, an immunomodulatory compound and dexamethasoneare administered as maintenance therapy to patients with multiplemyeloma following the transplantation of autologous bone marrow.

In another embodiment, an immunomodulatory compound is administeredfollowing the administration of high dose of melphalan and thetransplantation of autologous stem cell to patients with chemotherapyresponsive multiple myeloma.

In another embodiment, an immunomodulatory compound and PEG INTRO-A areadministered as maintenance therapy to patients with multiple myelomafollowing the transplantation of autologous CD34-selected peripheralstem cell.

In another embodiment, an immunomodulatory compound is administered withpost transplant consolidation chemotherapy to patients with newlydiagnosed multiple myeloma to evaluate anti-angiogenesis.

In another embodiment, an immunomodulatory compound and dexamethasoneare administered as maintenance therapy after DCEP consolidation,following the treatment with high dose of melphalan and thetransplantation of peripheral blood stem cell to 65 years of age orolder patients with multiple myeloma.

5.3.3 Cycling Therapy

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a patient. Cycling therapyinvolves the administration of an active agent for a period of time,followed by a rest for a period of time, and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid or reduce the side effects of oneof the therapies, and/or improves the efficacy of the treatment.

Consequently, in one specific embodiment of the invention, animmunomodulatory compound of the invention is administered daily in asingle or divided doses in a four to six week cycle with a rest periodof about a week or two weeks. The invention further allows thefrequency, number, and length of dosing cycles to be increased. Thus,another specific embodiment of the invention encompasses theadministration of an immunomodulatory compound of the invention for morecycles than are typical when it is administered alone. In yet anotherspecific embodiment of the invention, an immunomodulatory compound ofthe invention is administered for a greater number of cycles that wouldtypically cause dose-limiting toxicity in a patient to whom a secondactive ingredient is not also being administered.

In one embodiment, an immunomodulatory compound of the invention isadministered daily and continuously for three or four weeks at a dose offrom about 0.1 to about 150 mg/d followed by a break of one or twoweeks. Actimid™ is preferably administered daily and continuously at aninitial dose of 0.1 to 5 mg/d with dose escalation (every week) by 1 to10 mg/d to a maximum dose of 50 mg/d for as long as therapy istolerated. In a particular embodiment, Revimid™ is administered in anamount of about 5, 10, or 25 mg/day, preferably in an amount of about 10mg/day for three to four weeks, followed by one week or two weeks ofrest in a four or six week cycle.

In one embodiment of the invention, an immunomodulatory compound of theinvention and a second active ingredient are administered orally, withadministration of an immunomodulatory compound of the inventionoccurring 30 to 60 minutes prior to a second active ingredient, during acycle of four to six weeks. In another embodiment of the invention, thecombination of an immunomodulatory compound of the invention and asecond active ingredient is administered by intravenous infusion overabout 90 minutes every cycle. In a specific embodiment, one cyclecomprises the administration of from about 10 to about 25 mg/day ofRevimid™ and from about 50 to about 200 mg/m²/day of a second activeingredient daily for three to four weeks and then one or two weeks ofrest. In another specific embodiment, each cycle comprises theadministration of from about 5 to about 10 mg/day of Actimid™ and fromabout 50 to about 200 mg/m²/day of a second active ingredient for 3 to 4weeks followed by one or two weeks of rest. Typically, the number ofcycles during which the combinatorial treatment is administered to apatient will be from about one to about 24 cycles, more typically fromabout two to about 16 cycles, and even more typically from about four toabout three cycles.

5.4 Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions can be used in the preparation ofindividual, single unit dosage forms. Pharmaceutical compositions anddosage forms of the invention comprise an immunomodulatory compound ofthe invention, or a pharmaceutically acceptable salt, solvate, hydrate,stereoisomer, clathrate, or prodrug thereof. Pharmaceutical compositionsand dosage forms of the invention can further comprise one or moreexcipients.

Pharmaceutical compositions and dosage forms of the invention can alsocomprise one or more additional active ingredients. Consequently,pharmaceutical compositions and dosage forms of the invention comprisethe active ingredients disclosed herein (e.g., an immunomodulatorycompound and a second active agent). Examples of optional second, oradditional, active ingredients are disclosed herein (see, e.g., section5.2).

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), topical (e.g., eye drops or other ophthalmicpreparations), transdermal or transcutaneous administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; powders;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; eye drops or other ophthalmic preparations suitable fortopical administration; and sterile solids (e.g., crystalline oramorphous solids) that can be reconstituted to provide liquid dosageforms suitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms encompassedby this invention will vary from one another will be readily apparent tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition. Consequently, this invention encompasses pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

Lactose-free compositions of the invention can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmacopeia (USP) 25-NF20 (2002). In general, lactose-free compositionscomprise active ingredients, a binder/filler, and a lubricant inpharmaceutically compatible and pharmaceutically acceptable amounts.Preferred lactose-free dosage forms comprise active ingredients,microcrystalline cellulose, pre-gelatinized starch, and magnesiumstearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers”, include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise animmunomodulatory compound of the invention or a pharmaceuticallyacceptable salt, solvate, hydrate, stereoisomer, clathrate, or prodrugthereof in an amount of from about 0.10 to about 150 mg. Typical dosageforms comprise an immunomodulatory compound of the invention or apharmaceutically acceptable salt, solvate, hydrate, stereoisomer,clathrate, or prodrug thereof in an amount of about 0.1, 1, 2, 5, 7.5,10, 12.5, 15, 17.5, 20, 25, 50, 100, 150 or 200 mg. In a particularembodiment, a preferred dosage form comprises4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™) inan amount of about 1, 2, 5, 10, 25 or 50 mg. In a specific embodiment, apreferred dosage form comprises3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) in an amount of about 5, 10, 25 or 50 mg. Typical dosageforms comprise the second active ingredient in an amount of 1 to about1000 mg, from about 5 to about 500 mg, from about 10 to about 350 mg, orfrom about 50 to about 200 mg. Of course, the specific amount of theanti-cancer drug will depend on the specific agent used, the type ofcancer being treated or managed, and the amount(s) of animmunomodulatory compound of the invention and any optional additionalactive agents concurrently administered to the patient.

5.4.1 Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredients in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration. For example, excipients suitablefor use in oral liquid or aerosol dosage forms include, but are notlimited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or lowmoisture excipients or additives include AVICEL-PH-103™ and Starch 1500LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof. If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

A preferred solid oral dosage form of the invention comprises animmunomodulatory compound of the invention, anhydrous lactose,microcrystalline cellulose, polyvinylpyrrolidone, stearic acid,colloidal anhydrous silica, and gelatin.

5.4.2 Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

5.4.3 Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention. For example, cyclodextrin andits derivatives can be used to increase the solubility of animmunomodulatory compound of the invention and its derivatives. See,e.g., U.S. Pat. No. 5,134,127, which is incorporated herein byreference.

5.4.4 Topical and Mucosal Dosage Forms

Topical and mucosal dosage forms of the invention include, but are notlimited to, sprays, aerosols, solutions, emulsions, suspensions, eyedrops or other ophthalmic preparations, or other forms known to one ofskill in the art. See, e.g., Remington's Pharmaceutical Sciences,16^(th) and 18^(th) eds., Mack Publishing, Easton Pa. (1980 & 1990); andIntroduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,Philadelphia (1985). Dosage forms suitable for treating mucosal tissueswithin the oral cavity can be formulated as mouthwashes or as oral gels.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide topical and mucosal dosage forms encompassedby this invention are well known to those skilled in the pharmaceuticalarts, and depend on the particular tissue to which a givenpharmaceutical composition or dosage form will be applied. With thatfact in mind, typical excipients include, but are not limited to, water,acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol,isopropyl myristate, isopropyl palmitate, mineral oil, and mixturesthereof to form solutions, emulsions or gels, which are non-toxic andpharmaceutically acceptable. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well known in the art. See,e.g., Remington's Pharmaceutical Sciences, 16^(th) and 18^(th) eds.,Mack Publishing, Easton Pa. (1980 & 1990).

The pH of a pharmaceutical composition or dosage form may also beadjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

5.4.5 Kits

Typically, active ingredients of the invention are preferably notadministered to a patient at the same time or by the same route ofadministration. This invention therefore encompasses kits which, whenused by the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

A typical kit of the invention comprises a dosage form of animmunomodulatory compound of the invention, or a pharmaceuticallyacceptable salt salt, solvate, hydrate, stereoisomer, prodrug, orclathrate thereof. Kits encompassed by this invention can furthercomprise additional active ingredients such as oblimersen (Genasense®),melphalan, G-CSF, GM-CSF, EPO, topotecan, dacarbazine, irinotecan,taxotere, IFN, COX-2 inhibitor, pentoxifylline, ciprofloxacin,dexamethasone, IL2, IL8, IL18, Ara-C, vinorelbine, isotretinoin, 13cis-retinoic acid, or a pharmacologically active mutant or derivativethereof, or a combination thereof. Examples of the additional activeingredients include, but are not limited to, those disclosed herein(see, e.g., section 5.2).

Kits of the invention can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits of the invention can further comprise cells or blood fortransplantation as well as pharmaceutically acceptable vehicles that canbe used to administer one or more active ingredients. For example, if anactive ingredient is provided in a solid form that must be reconstitutedfor parenteral administration, the kit can comprise a sealed containerof a suitable vehicle in which the active ingredient can be dissolved toform a particulate-free sterile solution that is suitable for parenteraladministration. Examples of pharmaceutically acceptable vehiclesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

6. EXAMPLES

Certain embodiments of the invention are illustrated by the followingnon-limiting examples.

6.1 Modulation of Cytokine Production

A series of non-clinical pharmacology and toxicology studies have beenperformed to support the clinical evaluation of an immunomodulatorycompound of the invention in human subjects. These studies wereperformed in accordance with internationally recognized guidelines forstudy design and in compliance with the requirements of Good LaboratoryPractice (GLP), unless otherwise noted.

Inhibition of TNF-α production following LPS-stimulation of human PBMCand human whole blood by4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™),3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione andthalidomide (Revimid™) was investigated in vitro (Muller et al., Bioorg.Med. Chem. Lett. 9:1625-1630, 1999). The IC₅₀'s of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione forinhibiting production of TNF-α following LPS-stimulation of PBMC andhuman whole blood were ˜24 nM (6.55 ng/mL) and ˜25 nM (6.83 ng/mL),respectively. In vitro studies suggest a pharmacological activityprofile for3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione that issimilar to, but at least 200 times more potent than, thalidomide. Invitro studies have also demonstrated that concentrations of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione of 2.73 to27.3 ng/mL (0.01 to 0.1 μM) achieved 50% inhibition of the proliferationof MM.IS and Hs Sultan cells.

The IC₅₀'s of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione forinhibiting production of TNF-α following LPS-stimulation of PBMC andhuman whole blood were ˜100 nM (25.9 ng/mL) and ˜480 nM (103.6 ng/mL),respectively. Thalidomide, in contrast, had an IC₅₀ of ˜194 μM (50.2μg/mL) for inhibiting production of TNF-α following LPS-stimulation ofPBMC. In vitro studies suggest a pharmacological activity profile for3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione that issimilar to, but 50 to 2000 times more potent than, thalidomide. It hasbeen shown that the compound is approximately 50-100 times more potentthan thalidomide in stimulating the proliferation of T-cells followingprimary induction by T-cell receptor (TCR) activation.3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione is alsoapproximately 50 to 100 times more potent than thalidomide in augmentingthe production of IL-2 and IFN-γ following TCR activation of PBMC (IL-2)or T-cells (IFN-γ). In addition,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneexhibited dose-dependent inhibition of LPS-stimulated production of thepro-inflammatory cytokines TNF-α, IL-1β, and IL-6 by PBMC while itincreased production of the anti-inflammatory cytokine IL-10.

6.2 Inhibition of Mm Cell Proliferation

The ability of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) and thalidomide for comparison to effect the proliferation ofMM cell lines has been investigated in an in vitro study. Uptake[³H]-thymidine by different MM cell lines (MM.1S, Hs Sultan, U266 andRPMI-8226) was measured as an indicator of cell proliferation. Cellswere incubated in the presence of compounds for 48 hours; [³H]-thymidinewas included for the last 8 hours of the incubation period. Addition of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione toMM.1S and Hs Sultan cells resulted in 50% inhibition of cellproliferation at concentrations of 0.4 μm and 1 μm, respectively. Incontrast, addition of thalidomide at concentrations up to 100 μmresulted in only 15% and 20% inhibition of cell proliferation in MM.1Sand Hs Sultan cells, respectively. These data are summarized in FIG. 1.

6.3 Toxicology Studies

The effects of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) on cardiovascular and respiratory function are investigatedin anesthetized dogs. Two groups of Beagle dogs (2/sex/group) are used.One group receives three doses of vehicle only and the other receivesthree ascending doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione (2, 10,and 20 mg/kg). In all cases, doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione orvehicle are successively administered via infusion through the jugularvein separated by intervals of at least 30 minutes.

The cardiovascular and respiratory changes induced by3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione areminimal at all doses when compared to the vehicle control group. Theonly statistically significant difference between the vehicle andtreatment groups is a small increase in arterial blood pressure (from 94mmHg to 101 mmHg) following administration of the low dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Thiseffect lasts approximately 15 minutes and is not seen at higher doses.Deviations in femoral blood flow, respiratory parameters, and Qtcinterval are common to both the control and treated groups and are notconsidered treatment-related.

6.4 Cycling Therapy in Patients

In a specific embodiment, an immunomodulatory compound of the inventionare cyclically administered to patients with cancer. Cycling therapyinvolves the administration of a first agent for a period of time,followed by a rest for a period of time and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid or reduce the side effects of oneof the therapies, and/or improves the efficacy of the treatment.

In a specific embodiment, prophylactic or therapeutic agents areadministered in a cycle of about 4 to 6 weeks, about once or twice everyday. One cycle can comprise the administration of a therapeutic onprophylactic agent for three to four weeks and at least a week or twoweeks of rest. The number of cycles administered is from about one toabout 24 cycles, more typically from about two to about 16 cycles, andmore typically from about four to about eight cycles.

For example, in a cycle of four weeks, on day 1, the administration of25 mg/d of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione isstarted. On day 22, the administration of the compound is stopped for aweek of rest. On day 29, the administration of 25 mg/d3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidin-2,6-dione isbegun.

6.5 Clinical Studies in Patients

6.5.1 Treatment of Relapsed Multiple Myeloma

4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (Actimid™)was administered to patients with relapsed/refractory multiple myeloma.The study was conducted in compliance with Good Clinical Practices.Patients were at least 18 years old, had been diagnosed with multiplemyeloma (with paraprotein in serum and/or urine), and were consideredrefractory to treatment after at least two cycles of treatment, or haverelapsed after two cycles of treatment.

Patients who have progressive disease, according to the SouthwestOncology Group (SWOG) criteria, on their prior regimen are consideredtreatment refractory. Relapse following remission is defined as >25%increase in M component from baseline levels; reappearance of the Mparaprotein that had previously disappeared; or a definite increase inthe size and number of lytic bone lesions recognized on radiographs.Patients may have had prior therapy with thalidomide, provided they wereable to tolerate the treatment. A Zubrod performance status of 0 to 2 isrequired for all patients.

4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione isadministered to patients at doses of 1, 2, 5, or 10 mg/day for up tofour weeks; at each dose level, three patients are initially enrolled.Dosing occurs at approximately the same time each morning; all doses areadministered in the fasted state (no eating for at least two hours priorto dosing and two hours after dosing).4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione doses areadministered in an ascending fashion such that patients in the firstcohort receive the lowest dose of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (1 mg/day)and escalation to the next higher dose level occurs only following theestablishment of safety and tolerability at the current dose. If one outof three patients at any dose level experience dose limiting toxicity(DLT), three additional patients are enrolled at that dose. If none ofthe three additional patients experience DLT, escalation to the nextdose level occurs; dose escalations continue in a similar fashion untilthe MTD is established or the maximum daily dose (10 mg/day) isattained. However, if one of the three additional patients enrolledexperiences DLT, the MTD has been reached. If two or more of the threeadditional patients enrolled experience DLT, the MTD is judged to havebeen exceeded and three additional patients are enrolled at thepreceding dose level to confirm the MTD. Once the MTD has beenidentified, four additional patients are enrolled at that dose level sothat a total of 10 patients is treated at the MTD.

Blood sampling for analysis of pharmacokinetic parameters is performedon Days 1 and 28 according to the following sampling schedule: pre-dose,0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, and 24 hourspost-dose. An additional blood sample is collected at each weekly visitfor the determination of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione levels. Totalurine collections are also made with urine pooled according to thefollowing time intervals post-dose: 0 to 4, 4 to 8, 8 to 12, and 12 to24 hours. Safety assessments are made by monitoring adverse events,vital signs, ECGs, clinical laboratory evaluations (blood chemistry,hematology, lymphocyte phenotyping, and urinalysis), and physicalexamination at specific times during the study.

Results of interim pharmacokinetic analyses obtained following single-and multiple-dose administration of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione to multiplemyeloma patients are presented below in Tables 1 and 2. These data showthat 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione wassteadily absorbed at all dose levels in relapsed multiple myelomapatients. Maximum plasma concentrations occurred at a median T_(max) ofbetween 2.5 and 2.8 hours post-dose at Day 1 and between 3 and 4 hourspost-dose at Week 4. At all doses, plasma concentrations declined in amonophasic manner after reaching C_(max). The start of the eliminationphase occurred between 3 and 10 hours post-dose at Day 1 and Week 4,respectively.

These data also showed that after 4 weeks of dosing,4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione accumulatedto a small extent (mean accumulation ratios ˜1.02 to 1.52 and ˜0.94 to1.62 for C_(max) and AUC_((0-τ)), respectively). There was almost a doseproportional increase in AUC_((0-τ)) and C_(max) values with increasingdose. A five-fold higher dose of4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione produced a3.2- and 2.2-fold increase in C_(max) at Day 1 and Week 4, respectively.Similarly, a 5-fold increase in dose resulted in a 3.6- and 2.3-foldincrease in AUC_((0-τ)), at Day 1 and Week 4, respectively.

TABLE 1 Pharmacokinetic parameters of Actimid ™ in relapsed multiplemyeloma patients Parameter 1 mg (N = 6) 2 mg (N = 2) 5 mg (N = 3) Day 1C_(max) ng/mL 15.03 (4.04) 24.4* (12.1) 48.56 (14.03) t_(max) h 3.3(2.6) 2.7* (0.3) 2.3 (0.3) AUC_((0-∞)) ng · h/mL 152.90 (36.62) 279.18(51.10) 593.10 (335.23) AUC_((0-τ)) 134.21 (27.14) 249.57 (29.26) 520.94(267.32) t½ h 7.3 (3.4) 6.3 (1.4) 6.5 (2.2) CL/F mL/ min 114.75 (29.20)121.43 (22.22) 182.31 (117.06) Vz/f L 69.55 (44.97) 65.31 (2.80) 87.24(22.61) τ = 24 hours N/A = not available

TABLE 2 Pharmacokinetic parameters of Actimid ™ following multiple oraldoses(1, 2, and 5 mg/day) in relapsed multiple myeloma patientsParameter 1 mg (N = 5) 2 mg (N = 2) 5 mg (N = 3) Week 4 C_(max) ng/mL23.20 (7.48) 30.05* (15.64) 58.07 (38.08) t_(max) h 3.6 (1.5) 2.8* (0.3)5.0 (2.6) AUC_((0-∞)) ng · h/mL N/A N/A N/A AUC_((0-τ)) 239.31 (122.59)269.36 (186.34) 597.24 (354.23) t½ h 6.2* (0.6) 7.7 (2.8) 7.8 (4.0) CL/FmL/min 87.85 (48.48) 162.68 (112.54) 207.50 (175.41) Vz/f L 41.35*(8.84) 95.04 (35.39) 103.95 (27.25) τ = 24 hours N/A = not available *N= 3 patients

6.5.2 Treatment of Relapsed Multiple Myeloma

Two Phase 1 clinical studies of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) have been conducted to identify the maximum tolerated dose(MTD) in patients with refractory or relapsed multiple myeloma. Thesestudies have also characterized the safety profile of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione whenascending doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione weregiven orally for up to 4 weeks. Patients started3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionetreatment at 5 mg/day with subsequent escalation to 10, 25, and 50mg/day. Patients were enrolled for 28 days at their assigned dose, withthe option of extended treatment for those who did not exhibit diseaseprogression or experience dose limiting toxicity (DLT). Patients wereevaluated for adverse events at each visit and the severity of theseevents was graded according to the National Cancer Institute (NCI)Common Toxicity Criteria. Patients were discontinued if they experiencedDLT (Grade 3 or greater non-hematological, or Grade 4 hematologicaltoxicity).

In this study, 27 patients were enrolled. All patients had relapsedmultiple myeloma and 18 (72%) were refractory to salvage therapy. Amongthese patients, 15 had undergone prior autologous stem celltransplantation and 16 patients had received prior thalidomidetreatment. The median number of prior regimens was 3 (range 2 to 6).

Blood and urine samples were collected for analysis of pharmacokineticparameters on Days 1 and 28. Blood samples were collected according tothe following sampling schedule: pre-dose, 0.25, 0.5, 0.75, 1, 1.5, 2,2.5, 3, 4, 6, 8, 10, 12, 18, and 24 hours post-dose. In addition, ablood sample was collected at each weekly clinic visit for3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionedetermination. Total urine was collected and pooled according to thefollowing time intervals post-dose: 0 to 4, 4 to 8, 8 to 12, and 12 to24 hours. Response to treatment was assessed by M-protein quantification(by immunoelectrophoresis) from serum and a 24-hour urine collection,with creatinine clearance and 24-hour protein calculations undertaken atscreening, baseline, Weeks 2 and 4, and monthly thereafter (or uponearly termination). Bone marrow aspirations and/or tissue biopsy arealso performed at Months 3, 6 and 12 if a patient's paraprotein serumconcentration or 24-hour urine protein excretion declined to the nextlower level, based on best response criteria. Preliminary results forthe 28-day treatment period are summarized below.

Preliminary pharmacokinetic analyses based on these two studiesindicated that AUC and C_(max) values increase proportionally with dosefollowing single and multiple doses in multiple myeloma patients (as wasseen in healthy volunteers). Further, there was no evidence ofaccumulation with multiple dosing as single dose AUC_((0-∞)) wascomparable to multiple dose AUC_(0-τ), following the same dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione.Similar to healthy volunteer studies, double peaks were observed.Exposure in multiple myeloma patients appeared to be slightly higherbased on C_(max) and AUC values as compared to healthy male volunteerswhile clearance in multiple myeloma patients was lower than it was inhealthy volunteers, consistent with their poorer renal function (both asa consequence of their age and their disease). Finally,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionehalf-live in patients was shorter than in healthy volunteers (mean 8hours, ranging up to 17 hours).

In this study, the first cohort of 3 patients was treated for 28 days at5 mg/day without any dose limiting toxicity (DLT). The second cohort of3 patients subsequently commenced therapy at 10 mg/day. Patients in thesecond 10 mg/day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione cohorttolerated treatment well.

6.5.3 Treatment of Solid Tumors

Study with3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revimid™) was conducted in patients with varying types of solid tumors,including malignant melanoma (13), carcinoma of the pancreas (2),carcinoid-unknown primary (1), renal carcinoma (1), breast carcinoma (1)and NSCLC (2). Patients received 5 mg/day3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione forseven days and are subsequently escalated every seven days to 10 mg/day,25 mg/day, and 50 mg/day for a total of 4 weeks of treatment. Patientswho, experienced clinical benefit were permitted to continue ontreatment as Named Patients.

The study initially enrolled 20 patients and was subsequently amended toenroll 16 additional patients (adrenal carcinoma, NSCLC, malignantmesothelioma, breast cancer, malignant melanoma (8), renal cell cancer(4)) at a higher dose. The 16 additional patients were given weeklyescalating doses of 25 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 125mg/day, and 150 mg/day over a 6-week period with continuing treatmentfor an additional six weeks.

The study of Phase 1 study was designed to determine a maximum tolerateddose (MTD) of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione inpatients with refractory solid tumors and/or lymphoma, as well as tocharacterize the pharmacokinetic and side effect profiles of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione in thispatient population. The study design dictates that at least 3 patientsmust be enrolled at a dose level and have completed 28 days of treatmentprior to enrollment of patients at the next higher dose level. Patientsin the first cohort began dosing at 5 mg/day of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione.Patients will be escalated to 10, 20, 25, and 30 mg/day provided thereis no toxicity.

In this study, the MTD is defined as the highest dose level in whichfewer than two of six patients treated did not experience Grade 3 orgreater non-hematological toxicity or Grade 4 or greater hematologicaltoxicity. If, at any given dose level in either study, one out of threepatients experiences toxicity, three additional patients must be treatedat that particular dose. If, however, two out of six patients experienceDLT, the MTD is judged to have been exceeded. No further doseescalations are to occur and additional patients are to be enrolled atthe previous dose level. The dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneadministered is escalated until the MTD is achieved or the maximum dailydose of is reached.

No DLTs were reported in the initial group of 20 patients enrolled inthe study. Thirteen of the original 20 trial patients, along with 2non-trial patients, continued on treatment as named patients at doses upto 150 mg/day.

6.5.4 Treatment of Gliomas

This study was performed to find toxicity in patients with recurrent,high-grade gliomas. The study is designed such that patients are givenincreasingly higher doses of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione until amaximum tolerated dose (MTD) is established. The study also seeks toobtain preliminary toxicity information and pharmacokinetic data on3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione, aswell as to develop exploratory data concerning surrogate end points ofangiogenic activity in vivo using functional neuro-imaging studies, andin vitro assays of serum angiogenic peptides.

Patients enrolled in the first cohort receive 2.5 mg/m²/day for a 4-weekcycle. During each 4-week cycle of therapy,3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dioneadministered once daily for 3 weeks followed by a week of rest. Patientswho complete a treatment cycle may receive another cycle of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionetreatment if two criteria are met. First, the patient must have stabledisease or have experienced a partial response or complete response, orthe patient is benefiting from the therapy with3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione asevidenced by a decrease in tumor-related symptoms such as neurologicaldeficits. Second, the patient must have recovered from toxicity relatedto 3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dionewhich occurred in the prior cycle by Day 42 or sooner (28-day cycle pluslimit of 2 weeks to recover) as evidenced by a return to Grade≦1toxicity level. Patients who experience DLT in the previous cycle shouldhave their dose modified. DLT is defined as an non-hematological eventGrade≧3 toxicity or hematological event of Grade 4 toxicity thought tobe related to the study medication. Patients who experience DLT in thefirst cycle and have no response to therapy are removed from the study.

3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione dosesare subsequently escalated to 5, 8, 11, 15, and 20 mg/m²/day to amaximum total daily dose of 40 mg. Patients continue to receive3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione on a4-week cycle per dose level until one of the off-study criteria are met.

Three patients are enrolled in each cohort. If at least one DLT occurs,three additional patients are added to the cohort at that particulardose level. If two DLTs occur, the MTD, defined as the dose at whichfewer than one-third of patients at each dose level experiences DLT hasbeen exceeded and four more patients are treated at the previous dose.

Patients who experience DLT during the first 4-week cycle are removedfrom the study, except if they have a response to therapy. For patientswho have completed their first 4-week cycle of without DLT, but whosubsequently experience Grade 3 or 4 hematological and/ornonhematological toxicity, treatment is suspended for a minimum of aweek. If the toxicity resolves to <Grade 2 within three weeks, thepatient is treated at two dose levels lower than the dose that causedthe toxicity (or a 50% reduction if the patient was treated at the firstor second dose level). Patients in whom Grade 3 or 4 toxicity does notresolve to <Grade 1 within three weeks, or those who have another Grade3 toxicity at the reduced dose are removed from the study.

Pharmacokinetic sampling is performed prior the first dose of3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione (Day 1)and 0.5, 1, 2, 4, 6, 8, 24, and 48 hours thereafter. Sampling is alsoconducted pre-dose on Days 7 and 21 and 0.5, 1, 2, 4, 6, 8, and 24post-dose on Day 21 to evaluate steady-state3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione levels.

6.5.5 Treatment of Metastatic Melanoma

Patients with metastatic melanoma were started on3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione(Revmid™) at 5 mg/day for seven days. The dose was then increased everyseven days to 10 mg/day, 25 mg/day, and 50 mg/day, respectively, for atotal of four weeks on therapy. Five of the 13 melanoma patients whowere treated under this regimen either showed disease stabilization or apartial response in the first four weeks of treatment. Tumor responsewas seen in cutaneous and subcutaneous lesions (five patients), lymphnodes (two patients), and liver (one patient). The duration of responsewas approximately six months. The result suggests that the compoundappears is a promising new anti-cancer agent and has both antiangiogenicand immunomodulatory properties.

6.5.6 Treatment of Relapsed or Refractory Multiple Myeloma

Patients with relapsed and refractory Dune-Salmon stage III multiplemyeloma, who have either failed at least three previous regimens orpresented with poor performance status, neutropenia or thrombocytopenia,are treated with up to four cycles of combination of melphalan (50 mgintravenously), an immunomodulatory compound of the invention (about 1to 150 mg orally daily), and dexamethasone (40 mg/day orally on days 1to 4) every four to six weeks. Maintenance treatment consisting of dailyan immunomodulatory compound of the invention and monthly dexamethasoneare continued until the disease progression. The therapy using animmunomodulatory compound of the invention in combination with melphalanand dexamethasone is highly active and generally tolerated in heavilypretreated multiple myeloma patients whose prognosis is otherwise poor.

The embodiments of the invention described above are intended to bemerely exemplary, and those skilled in the art will recognize, or willbe able to ascertain using no more than routine experimentation,numerous equivalents of specific compounds, materials, and procedures.All such equivalents are considered to be within the scope of theinvention and are encompassed by the appended claims.

What is claimed is:
 1. A method of treating hepatocellular carcinoma,which comprises administering to a patient having hepatocellularcarcinoma about 5 to about 25 mg per day of a compound3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione of theformula:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.2. The method of claim 1, wherein the hepatocellular carcinoma ismetastatic hepatocellular carcinoma, unrescectable hepatocellularcarcinoma, refractory hepatocellular carcinoma, or relapsedhepatocellular carcinoma.
 3. The method of claim 1, wherein the compoundis


4. The method of claim 1, wherein the compound is a pharmaceuticallyacceptable salt.
 5. The method of claim 1, wherein the compound is apharmaceutically acceptable solvate.
 6. The method of claim 1, whereinthe compound is a pharmaceutically acceptable stereoisomer.
 7. Themethod of claim 6, wherein the stereoisomer is an enantiomerically pureR isomer.
 8. The method of claim 6, wherein the stereoisomer is anenantiomerically pure S isomer.
 9. The method of claim 1, which furthercomprises administering a therapeutically effective amount of a secondactive agent.
 10. The method of claim 9, wherein the second active agentis hematopoietic growth factor, a cytokine, or an anti-cancer agent. 11.The method of claim 9, wherein the second active agent is interferonalpha, capecitabine, cisplatin. thiotepa, foxorubicin, sorafenib,cetuximab, gemcitabine, bevacizumab, oxaliplatin, temserolimus,everolimus, ifosfamide, carboplatin, etoposide, 5-fluorouracil,vincristine, cyclophosphamide, epirubicin, paclitaxel. octreotide,geftinib, erlotinib, sunitinib, cediranib, bortezomib, azacitidine,amrubicin, anthracycline, or a combination thereof.
 12. The method ofclaim 1, wherein the hepatocellular carcinoma is relapsed. refractory orresistant to conventional therapy.
 13. The method of claim 1, whereinthe compound is administered orally.
 14. The method of claim 13, whereinthe compound is administered in the form of a capsule or tablet.
 15. Themethod of claim 1, wherein the compound is administered in an amount offrom about 10 to about 25 mg per day.
 16. The method of claim 14,wherein the compound is administered in an amount of about 2.5, 5, 10,15, 20, or 25 per day.
 17. The method of claim 1, wherein the compoundis administered in an amount of about 25 mg per day.
 18. The method ofclaim 1, wherein the compound is administered cyclically.
 19. The methodof claim 18, wherein one cycle comprises four to six weeks.
 20. Themethod of claim 18, wherein one cycle comprises the administration ofthe compound for 21 days followed by seven days rest.
 21. The method ofclaim 18, wherein the compound is administered for four to twenty-fourweeks with one to six weeks of rest.
 22. The method of claim 1, whereinthe compound is administered in an amount of from about 5 to about 25 mgper day for days 1-21 every 28 days.
 23. The method of claim 18, whereinthe compound is administered in an amount of about 25 mg per day for 21days followed by seven days rest in a 28 day cycle.
 24. The method ofclaim 1, wherein the compound is administered in an amount of about 5 mgper day.
 25. The method of claim 1, wherein the compound is administeredin an amount of 10 mg per day.
 26. The method of claim 1, wherein thecompound is administered in an amount of 15 mg per day.
 27. The methodof claim 1, wherein the compound is administered in a capsule comprising5 mg, 10 mg, 15 mg or 25 mg of the compound or pharmaceuticallyacceptable salt, solvate or stereoisomer thereof.
 28. The method ofclaim 14, wherein the capsule comprises the compound, lactose anhydrous,microcrystalline cellulose, croscarmellose sodium and magnesiumstearate.